The formation of metastases from a primary tumor is a complex temporal process that involves angiogenesis, invasion of the circulatory system by tumor cells, intravasation of the endothelium, arrest in the venous or capillary bed of the target organ, extravasation, entry into the target parenchyme and proliferation of the secondary tumor in tissue different from its tissue of origin. Throughout this process metastatic tumor cells are constantly interacting with their host tissue modulating their adhesiveness to cells and extracellular matrices, degrading matrices and migrating into interstitial stroma. These events are not unique to tumor metastasis and occur in other processes such as angiogenesis, tissue remodeling, bone remodeling and embryogenesis. Experimental studies have demonstrated that invasion involves the active locomotion of tumor cells through and into tissue barriers. The molecular mechanisms regulating such invasive tumor cell migration and subsequent implantation are poorly understood but it appears that there is organ specificity of colonization.
Several substances that stimulate tumor cell locomotion in vitro have been described. These include factors derived from resorbing bone, liver and smooth muscle. Locomotion of tumor cells can also be induced by endogenous substances in an autocrine fashion. These factors can induce two types of directed cell locomotion: (1) chemotaxis, i.e., directed locomotion of cells up a soluble gradient; or (2) haptotaxis, i.e., migration of cells up a gradient of attached molecules.
Bone metastases are frequently one of the first signs of disseminated disease in certain carcinomas of lung, breast, prostate, kidney or thyroid. Osteoblasts, the bone forming cells, have been shown to secrete substances that induce the chemotaxis and invasion of melanoma and breast cancer cells. One product of osteoblasts that induces the migration of several cell types and is secreted by several tumors is osteopontin.
Osteopontin (Oldberg et al. (1986) Proc. Natl. Acad. Sci. USA 83:8819; Oldberg et al. (1986) J. Biol. Chem. 263:19433-19436) also known as OPN (Wrana et al. (1989) Nucl. Acid Res. 17:3306), 2ar (Smith, J. H. and Denhardt, D. T. (1987). J. Cell Biochem. 34: 10-22), transformation-associated secreted phosphoprotein (Senger et al. (1989) Anticancer Res. 48:1291), or Early T-lymphocyte activation-1 (Patarca et al. (1991) Proc. Natl. Acad, Sci. USA 88:2736), is a secreted glycosylated phosphoprotein expressed by bone (Oldberg et al. (1986) J. Biol. Chem. 263: 19433-19436), activated T-lymphocytes (Patarca et al. (1989) J. Exp. Med. 170:145-161; Patarca et al. (1991) Proc. Natl. Acad. Sci. USA 88:2736), macrophages (Singh et al. (1990). J. Exp. Med 171:1931-1942), smooth muscle cells of the vascular system (Giachelli et al. (1991) Biochem. Biophys. Res. Commun. 177: 867-873), and carcinomas and sarcomas (Senger et al. (1989) Anticancer Res. 48:1291).
The marked induction of osteopontin during arterial wound healing, immune response, and bone development and remodeling, suggests a role for this protein in these processes. Osteopontin expression by smooth muscle cells is induced upon arterial injury were it is chemotactic to smooth muscle cells and supports the adhesion of endothelial cells. Osteopontin is also abundant in athrosclerotic plaques. Secretion of osteopontin in the early response after T-cell activation is associated with enhanced secretion of IgM and IgG by B-cells (Lampe et al. (1991) J. Immunol. 147:2902) and is chemotactic to macrophages (Singh et al. (1989) Anticancer Res. 48:1291). It is constitutively expressed in CD4xe2x88x92CD8xe2x88x92T lymphocytes from the spontaneously autoimmune MRL/1pr mouse strain (Patarca et al. (1990) J. Exp. Med. 172:1177-1183). Its circulating levels are elevated in individuals with autoimmune diseases. Osteopontin is also involved in bone development and remodeling. Osteopontin supports the migration and adhesion of osteoclasts and osteoblasts and appears to be chemotactic to osteoprogenitor cells.
Osteopontin is also elevated in sera from patients with advanced metastatic cancer and cellular transformation may lead to enhanced osteopontin expression and increased metastatic activity. Expression of antisense RNA in metastatic Ras transformed fibroblasts resulted in the reduction of the metastatic potential of these cells. The presence of a Gly-Arg-Gly-Asp-Ser (GRGDS, SEQ ID NO:8) cell-surface receptor binding motif within the sequence of osteopontin suggested that osteopontin may be involved in cell attachment and spreading (Oldberg et al. (1986) Proc. Natl. Acad. Sci. USA 83:88 19; Oldberg et al. (1986) J. Biol. Chem. 263:19433-19436). Osteopontin binds to cells via integrin and non-integrin receptors, and is a ligand for xcex1vxcex23, xcex1vxcex21, and xcex1vxcex25 integrins. Multiple phosphorylated and nonphosphorylated forms of osteopontin are secreted by cells and are differentially stimulated by tumor promoters (Kubota et al. (1989) Biochem. Biophys. Res. Commun. 162: 1453-1459). In addition, differential attachment of osteoclasts to surfaces coated with osteopontin isolated from various tissues and to phosphorylated and nonphosphorylated osteopontin has been demonstrated. Furthermore, cleavage of osteopontin with thrombin enhances its cell attachment properties. These results suggest that depending on the cell surface receptor repertoire, cells may recognize distinct forms of osteopontin and may respond differently to the form of osteopontin they encounter.
The present invention is based, at least in part, on the discovery of or identification of the chemotactic regions of the osteopontin polypeptide. This discovery led to the development of chemotactic peptides derived from osteopontin. The peptides (or antagonists of the same) can be used to treat conditions or diseases associated with chemotaxis. These peptides can further be used to treat conditions or diseases which can be treated using osteopontin, e.g., based upon osteopontin""s chemotactic properties. For example, the peptides of the present invention can be used to treat or inhibit tumor metastasis, inflammation, osteoporosis and autoimmune disease.
The present invention pertains to osteopontin derived peptides. The peptides are capable of inducing the chemotaxis of several cell types. Examples of cell types include, but are not limited to, endothelial cells, periosteal cells, tumor cells, macrophages and osteoprogenitor cells. The osteopontin derived chemotactic peptides do not appear to mediate cell attachment but rather alter the cytoskeletal organization of the cell and induce migration.
The invention also pertains to an isolated nucleic acid encoding an osteopontin derived peptide(s) of the present invention. The nucleic acid can be used to produce the peptide and also as a therapeutic agent.
Other aspects of the invention include antibodies, e.g., monoclonal antibodies, which are specifically reactive with the above-described peptides. These antibodies can be administered to a subject in the form of a therapeutic composition to modulate the chemotactic effect of the peptides of the invention. The preferred antibody of the invention has the amino acid sequence KFHSHKDKLVLDPKSK (SEQ ID NO:2). The antibodies neutralize the migration of various cell types in response to osteopontin both in vitro and in vivo.
In another aspect, the invention features a therapeutic composition which includes an osteopontin derived chemotactic peptide and a pharmaceutically-acceptable carrier or diluent. The therapeutic composition can be used in the methods described herein.
In another aspect, the invention features a method for modulating tumor invasion in a subject. The method includes administering to a subject a therapeutically effective amount of an antagonist of an osteopontin derived chemotactic peptide such that tumor invasion is modulated.
In another aspect, the invention features a method for promoting wound healing in a subject. The method includes administering to a subject a therapeutically effective amount of a composition comprising an osteopontin derived chemotactic peptide and a pharmaceutically-acceptable carrier or diluent such that wound healing is promoted.
In another aspect, the invention features a method for modulating tumor metastasis formation. The method includes administering to a subject a therapeutically effective amount of an antibody specifically reactive with an osteopontin derived chemotactic peptide such that tumor metastasis formation is modulated.
In another aspect, the invention features a method for promoting cell migration to a target site. The method includes administering to a cell a therapeutically effective amount of an osteopontin derived chemotactic peptide such that migration of the cell to the target site is promoted.
In another aspect, the invention features a method for modulating cellular chemotaxis. The method includes administering to a cell a therapeutically effective amount of an osteopontin derived chemotactic peptide such that modulation of cellular chemotaxis occurs.
The invention also features a prosthetic device. The prosthetic device contains a therapeutically effective amount of an osteopontin derived chemotactic peptide in the prosthetic device.
In another aspect, the invention features a method for treating the formation of atherosclerotic plaques. The method includes administering to a subject a therapeutically effective amount of an osteopontin derived chemotactic peptide such that formation of artherosclerotic plaques is prevented.
In another aspect, the invention also features a method for treating an angiogenic-associated disease. The method includes administering to a subject a therapeutically effective amount of an antibody specifically reactive with an osteopontin derived chemotactic peptide such that treatment of angiogenic-associated disease occurs.
In yet another aspect, the invention features a method of inducing either in vitro or in vivo chemotaxis of a cell. The method includes administering to a cell an osteopontin derived chemotactic peptide in an amount effective to induce chemotaxis.
Other aspects of the invention include methods for inducing cell migration. The method generally includes contacting the cell, e.g., a cell involved in wound healing or a cell, e.g., a neoplastic cell, the movement of which it is desirable to control, with the osteopontin derived chemotactic peptide of the invention. The antibodies specifically reactive with the osteopontin derived peptides of the invention or antagonists thereof can also be administered to a subject having a metastatic disease, e.g., cancer, to modulate tumor invasion, e.g., to prevent or inhibit metastasis of the disease by inhibiting the chemotactic activity of osteopontin. The peptides and antibodies can be administered to the subject in the form of a therapeutic composition which includes the peptide or antibody and a pharmaceutically acceptable carrier or diluent.
The osteopontin derived chemotactic peptides and methods of the present invention are useful for modulating chemotactic activity of osteopontin. For example, osteopontin derived chemotactic peptide are useful for stimulating chemotactic activity of osteopontin. Antagonists and antibodies raised against the peptides of the invention are useful for decreasing and/or inhibiting chemotactic activity of osteopontin.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.